Newly synthesized eukaryotic mRNA molecules, also known as primary transcripts or pre-mRNA, made in the nucleus, are processed before or during transport to the cytoplasm for translation. Processing of the pre-mRNAs includes addition of a 5′ methylated cap and an approximately 200-250 nucleotides poly(A) tail to the 3′ end of the transcript.
Another step in mRNA processing is splicing of the pre-mRNA, which is part of the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of a primary transcript that are not included in the coding sequence of the mature mRNA. Exons are regions of a primary transcript that remain in the mature mRNA when it reaches the cytoplasm. The exons are spliced together to form the mature mRNA sequence. Splice junctions are also referred to as splice sites with the junction at the 5′ end of the intron often called the “5′ splice site,” or “splice donor site” and the junction at the 3′ end of the intron called the “3′ splice site” or “splice acceptor site.” In splicing, the 3′ end of an upstream exon is joined to the 5′ end of the downstream exon. Thus the unspliced RNA (or pre-mRNA) has an exon/intron junction at the 5′ end of an intron and an intron/exon junction at the 3′ end of an intron. After the intron is removed, the exons are contiguous at what is sometimes referred to as the exon/exon junction or boundary in the mature mRNA. Alternative splicing, defined as the splicing together of different combinations of exons or exon segments, often results in multiple mature mRNA transcripts expressed from a single gene.
The splicing of precursor mRNA (pre-mRNA) is an essential step in eukaryotic gene expression, where introns are removed through the activities of the spliceosome, and the coding parts of a gene are spliced together, resulting in a functional mRNA. Pre-mRNA splicing is a highly controlled process and it is well established that mutations can impact splicing and generate aberrant transcripts [Andresen and Krainer 2009; Adkin et al. 2012, Olsen et al. 2014]. Correct mRNA splicing depends on regulatory sequences, which are recognized by different factors of the spliceosome, as well as splicing regulatory factors. The splicing regulatory factors either stimulate or repress recognition and splicing of exons by sequence specific binding to splicing regulatory sequences such as splicing enhancers and splicing silencers [Divina et al. 2009]. Pre-mRNA splicing in eukaryotes is often associated with extensive alternative splicing to enrich their proteome [Black 2000]. Alternative selection of splice sites permits eukaryotes to modulate cell type specific gene expression, contributing to their functional diversification. Alternative splicing is a highly regulated process influenced by the splicing regulatory proteins, such as SR proteins or hnRNPs, which recognize splicing regulatory sequences, such as exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs) in exons [Busch and Hertel 2012].
It is a well-known fact that exonic mutations, which either create or eliminate existing splicing regulatory sequences other than the splice site sequences often lead to missplicing of the RNA that might result in diseases. However, it is difficult to predict which mutations affect splicing as not all exons are critically dependent on splicing regulatory elements other than the splice sites, and consequently only a limited number of exons are vulnerable to mutations in splicing regulatory sequences outside of the splice sites [Andresen and Krainer 2009].
In recent years, a new class of genetic diseases has emerged that includes the clinically overlapping disorders Cardio-facio-cutaneous (CFC), Noonan and Costello syndromes (NS and CS, respectively). Even though genetic studies have revealed both molecular and clinical heterogeneity of these disorders, the common denominator is the association to the RAS-MAP kinase pathway and the class I phosphoinositide 3-kinase (PI3K) pathway [reviewed by Zenker 2011].
Costello syndrome is a rare inherited congenital disorder with a characteristic prenatal phenotype, caused by activating germline mutations in HRAS proto-oncogene [Aoki et al. 2005]. Costello syndrome belongs to a class of genetic syndromes that are caused by disorder of the RAS-MAP kinase pathway and the PI3K/Akt pathway. The HRAS protein is important in correct regulation of cell growth and division, and mutations in the HRAS gene might lead to numerous types of cancers, such as lung, skin, breast and colon. It has been observed in Costello patients as well as sporadic cancers that mutations in exon 2 of the gene, leads to a constitutive active HRAS protein, loss of cell cycle control and development of cancer.
Costello syndrome is usually caused by dominant negative germline mutations in HRAS exon 2, changing the codon for Glycine 12 or Glycine 13 to other amino acids. Such mutations results in a constitutive active Ras protein and activation of the Ras/MAPK and PI3K/Akt pathways, causing multiple developmental defects and a predisposition to cancer. Interestingly, the vast majority of somatic HRAS mutations in cancers change the codon for Glycine 12 to Valine, with a c.35G>T mutation being extremely frequent. Glycine 12 to Valine (G12V) mutations are rare in Costello syndrome patients, and usually cause a very severe clinical phenotype when present.
The three closely related human RAS genes, HRAS, KRAS and NRAS are all widely expressed and are important for regulation of numerous cellular processes through the RAS-MAP-kinase and PI3K/Akt pathways. They each exhibit oncogenic activity and more than 30% of all human tumors have mutations leading to constitutively active RAS proteins [Quinlan et al. 2008]. Different RAS oncogenes are preferentially associated with different types of human cancer [Parikh et al. 2007]. Therefore, the RAS oncogenes are already targets for numerous different anticancer treatments.
Knock down of expression from the dominant negative mutant HRAS allele does have enormous therapeutic potential for treating patients suffering from numerous types of cancers, and potentially also patients suffering from Costello syndrome and other Rasopathies.
Knock down of gene expression can be achieved by skipping of vulnerable exons during pre-mRNA splicing. This can be accomplished by using splice shifting oligonucleotides (SSOs) targeted to splicing regulatory signals, such as the splice sites or exon splicing enhancers (ESEs) which are fundamental for inclusion of weak exons (Kole et al. 2012). Exons which are weakly defined and thus difficult to splice often exhibit minimal levels of exon skipping also from wild type alleles. This type of vulnerable exons are preferred as targets for SSO mediated exon skipping (Fletcher et al. 2012).
SSOs have significant advances over existing therapeutic approaches: 1. SSOs target gene-specific sequences, which ensures that side-effects are minimal. 2. In sharp contrast to other antisense technologies SSOs are chemically modified to ensure superior long term stability and avoid degradation of the target mRNA. They can be further modified for enhanced cellular uptake and specific cancer cell targeting. 3. Contrary to RNAi, SSOs do not depend on the cellular RISC/RNase H or other cellular systems mediating mRNA degradation.
For efficient knock down by SSOs it is crucial that the targeted exon is weakly defined/vulnerable and thus critically dependent on a finely tuned balance between splicing enhancers and splicing silencers. Vulnerability is usually caused by weak splice sites and/or overrepresentation of exonic splicing silencers (ESSs) and/or underrepresentation of exonic splicing enhancers (ESEs) in the vulnerable exon.
SSO can mediate alternative splicing of the targeted pre-mRNA and thereby simultaneously lead to production of new protein isoforms with a dominant negative effect thereby further potentiating the effect of down regulation of the normal protein isoform.